Freshwater Crayfish 22(1):43–51, 2016 RESEARCH ARTICLE ISSN: 2076-4324 (Print), 2076-4332 (Online) https://doi.org/10.5869/fc.2016.v22-1.43

Ectosymbiotic Cleaners Induce Down-regulation of Crayfish Immune Response Genes

Bryan L. Brown,1,* Matthew Turnbull,2 James Skelton 3,5 and Robert P. Creed 4

1 Department of Biological Sciences, 2105 Derring Hall, Virginia Tech, Blacksburg, VA 24061 USA. *Corresponding Author.— [email protected] 2 Department of Biological Sciences, Clemson University, Clemson, SC 29631 USA. 3 School of Forest Resources and Conservation, University of Florida , Gainesville, FL 32601 USA. 4 Department of Biology, Appalachian State University, Boone, NC 28607 USA 5 Current Address: School of Forest Resources, University of Florida, Gainesville, Florida, 32601 USA.

ABSTRACT ARTICLE INFO Recent studies have revealed integral and complex relationships between hosts and their symbionts. Article History: Submitted: 02 JUL 2016 Some of these findings demonstrate that symbionts can affect the gene expression of their hosts. Accepted: 15 DEC 2016 We used a model cleaning symbiosis system of crayfish and their branchiobdellidan symbionts to Published Online: 31 DEC 2016 examine whether symbionts could affect host gene expression through indirect means, by changing the Published Print: 31 DEC 2016 interaction of the host crayfish with the environment. Previous research has shown this symbiosis to Keywords: be a complex, context dependent relationship in which outcomes can shift between mutualism, when crayfish; branchiobdellidans act as cleaners of their hosts, and parasitism, when damage to hosts’ gills through branchiobdellidan; branchiobdellidan feeding produces negative effects. These shifts are known to occur with changes in symbiosis; gene expression; symbiont densities and environmental conditions. We manipulated densities of branchiobdellidans on mutualism; host crayfish and examined the effect on expression of 3 candidate immune genes. We had 2 competing cleaning symbiosis; working hypotheses: 1) That branchiobdellidans would cause an increase expression in immune genes because of damage to the hosts’ gills; and 2) That branchiobdellidans would decrease expression of immune genes by acting as cleaners and thus alleviating environmental stress. We found that the second hypothesis was strongly supported when branchiobdellidan activity decreased expression in 2 of the 3 candidate genes. One of these genes (astacidin) is related to generalized immune defense, while the other (prophenoloxidase) is a key part of the melanization cascade that provides defense in the case of physical damage and intrusion of microbes or foreign bodies. Given that there is no known mechanism through which branchiobdellidans can directly affect host physiology through chemical or genetic interactions, we concluded that this alteration in gene expression occurred through alleviation of environmental stress by the symbionts. Such indirect effects may be common in nature and continued study using easily manipulated systems like the crayfish-branchiobdellidan symbiosis may continue to produce insights regarding the importance of symbioses in ecological systems.

Copyright © 2016 by The Author(s). Published by the International Association of Astacology. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION our early Egyptian pioneers would have ever imagined. We now The more we know about symbiosis, the more we appreciate know that symbionts can exert truly astonishing effects on their the ubiquity and importance of symbiotic interactions. While the hosts. Some of these relationships are quite complicated and even study of symbiosis dates back at least to the ancient Egyptians who bizarre, like fungi and parasitic worms that literally exert “mind included intestinal symbionts in early medical texts circa 1550 control” over their insect hosts (Thomas et al. 2002; Mongkolsamrit BCE (Egerton 2014), modern science has revealed that the biology et al. 2012). Even familiar and well-publicized symbioses like the of symbionts and hosts are often far more intimately entwined than relationships between plants and their pollinators, the influences of 43 44 Freshwater Crayfish Volume 22, Number 1 mycorrhizal symbioses, and the interdependence between corals thought to occur because branchiobdellidans clean crayfish body and their endosymbiotic dinoflagellates have proven to be far surfaces, particularly the gills, through their feeding behaviors, more complicated and varied than previously thought (Rodriguez- but can shift to direct consumption of host gill tissue when Lanetty et al. 2006; Karst et al. 2008; Carstensen et al. 2016). resources are limited (Brown et al. 2002; Brown et al. 2012). Recent work even suggests that our own evolutionary history, The relationship is context-dependent and can shift between current health, and survival as a species are intimately related to mutualism, commensalism, and parasitism based on a variety our microbial symbionts (Backhed et al. 2005; Yatsunenko et al. of factors including symbiont density (Brown et al. 2012), host 2012; Le Chatelier et al. 2013; Moeller et al. 2014). ontogeny (Thomas et al. 2016), and environmental conditions (Lee et al. 2009). Both host and symbiont can exert some influence One relatively recently discovered type of host-symbiont over the relationship, with hosts using grooming to remove excess interaction is the influence of symbionts on host gene expression. branchiobdellidans (Farrell et al. 2014; Skelton et al. 2014; Skelton These effects have been documented in a wide range ofhost- et al. 2016), and the branchiobdellidans showing evidence of host symbiont interactions, including bacteria and protozoans (Choi et choice (Brown and Creed 2004) and selective dispersal to optimize al. 1997), plants and mycorrhizal fungi (Zouari et al. 2014), and their own fitness (Skelton et al. 2015). bacteria that produce bioluminescence in several species of marine invertebrates (Almada and Tarrant 2016). We call these types of To examine the influence of branchiobdellidan symbionts interactions “recently discovered” because, even though they on host immune function, we conducted an experiment in which have been suspected for some time, only recently has technology we manipulated branchiobdellidan density on crayfish hosts, and allowed a thorough investigation of gene expression in real time. measured the expression of 3 candidate immune genes in the host To date, the majority of described cases in which symbionts affect crayfish during a 6-day experiment. In this experiment, we created the gene expression of hosts is through the symbiont directly conditions in which there was relatively high potential for microbial interacting with host physiology, for example, the influence of gill fouling of crayfish, a phenomenon that can be harmful or even Wolbachia on wasp hosts (Kremer et al. 2012), bacterial influence lethal to crustaceans (Bauer 1998), but in crayfish this fouling can over plant flowering (Lutay et al. 2016), arbuscular mycorrhizal be ameliorated to some degree by branchiobdellidans (Lee et al. fungi affecting immune defense of grapes against nematodes (Li et 2009). We also used a fairly high density of branchiobdellidans al. 2006), or the protozoan Trypanasoma promoting susceptibility as our experimental treatment so that significant gill damage to to transmission in mammals by altering expression in several the host was also a possibility (Brown et al. 2012). We therefore host pathways (Garcia-Silva et al. 2014). These previous studies had two competing working hypotheses regarding the effect of of symbiont effects on host gene expression mostly involve branchiobdellidans on host gene expression. 1) That we would endosymbionts, usually bacteria or fungi, that affect host gene measure decreased expression of immune-related genes in the expression through direct interactions with host physiology. branchiobdellidan treatments relative to the controls because the However, it is also probable that some types of symbionts can worms were alleviating gill fouling by cleaning. 2) That we would indirectly affect gene expression of their hosts by changing measure up-regulation of immune associated genes in response to the interaction between the host and environment. One type tissue damage caused by branchiobdellidans at fairly high densities. of symbiotic interaction in which these types of indirect effects Both of these hypotheses are compared to a null hypothesis of no might be expected are cleaning symbioses in which ectosymbionts effect of branchiobdellidans on host gene expression. remove parasites or foreign material from hosts, providing a beneficial cleaning service (Limbaugh 1961; Grutter et al. 2002). METHODS In these types of interactions, cleaners rarely interact with hosts in Laboratory Experiment such a way that host physiology is directly affected, but cleaners We conducted a 6-day aquarium experiment in June of can change the way that hosts interact with their environments, 2010 in which we manipulated branchiobdellidan density on 24 and thus could be expected to alter expression of host genes that host crayfish. The presence or absence of branchiobdellidans control a host’s interaction with their environment. was the only treatment in the experiment. We manipulated We investigated the potential for cleaners to indirectly induce branchiobdellidan presence by field collecting host crayfish, then changes in host gene expression using the cleaning symbiosis chemically killing their branchiobdellidan symbionts, and finally between freshwater crayfish (Arthropoda: ) and by adding branchiobdellidans back to the host at chosen treatment branchiobdellidans (Annelida: Oligochaeta). Branchiobdellidans levels using methods established during prior experiments (Brown are a group of ≈150 species of ectosymbiontic worms found on et al. 2002; Brown and Creed 2004; Brown et al. 2012). The host freshwater crustaceans in a Holarctic distribution (Gelder 1999). crayfish species was chasmodactylus James and the Until recently, the majority of branchiobdellidan species were branchiobdellidan symbiont was Cambarincola ingens Hoffman. considered to be commensals that had little effect on their hosts We field captured C. chasmodactylus from the South Fork of (Gelder 2010). However, a number of recent studies demonstrate the New River and selected 24 hosts that fell into a range of that the relationship between crayfish and branchiobdellidans can carapace lengths (CL) from 26.5 mm to 38.7 mm with a mean be multifaceted and complex. The relationship can shift between of 31.8mm. We chemically removed branchiobdellidans from a mutualism and parasitism, with worms sometimes promoting host crayfish using a 10 minute immersion in a 10% magnesium crayfish growth, but can also shift to parasitism under some chloride hexahydrate solution, a method demonstrated to kill conditions (Brown et al. 2002; Brown et al. 2012). This shift is the worms and their embryos contained in cocoons, but to have 2016 Brown et al. — Symbionts Influence Crayfish Gene Expression 45 little influence on crayfish hosts (Brown et al. 2002). Eachof the 24 hosts were assigned to one of 2 treatment levels, 0 or 10 branchiobdellidans. We then rinsed the crayfish in stream water and re-applied branchiobellidans (C. ingens) to the host using dissection probes (Brown et al. 2002; Brown et al. 2012). We chose 10 branchiobdellidans as our treatment because this density is high, but realistic for larger C. chasmodactylus (Brown and Creed 2004), and densities in this range have been demonstrated to have negative impacts on C. chasmodactylus (Brown et al. 2012). We watched each host with branchiobdellidans to ensure that the worms had attached and settled on to their hosts, then the crayfish were placed in aquaria. Prior to their placement in aquaria, half of each treatment group was randomly assigned to be collected at either 2-days or 6-days because sampling in this experiment was destructive, resulting in 6 replicate hosts for each treatment × time combination. Once assigned to treatments and the branchibodellidan manipulations had been performed, we placed crayfish in assigned aquaria for the remainder of the experiment with each crayfish assigned to an individual aquarium. Aquaria (37 L) were filled with water from the South Fork of the New River in Boone, NC. We collected the water from just below the Boone waste water treatment effluent discharge pipe. Our reasoning was that bacterial loads in the effluent water might be higher than normal river water which would potentially lead to higher bacterial loads on the crayfish gills thus promoting a greater immune response. Aquaria were aerated and contained 3-hole bricks that crayfish could use Figure 1. Branchiobdellidans (Xironodrilus appalachius) on the walking as refugia. Aquaria were placed on wire racks in a room in which legs of a crayfish host (Cambarus bartonii). The off-white spheres are water temperature ranged from 16 – 17°C. Lights in the room were branchiobdellidan cocoons, each of which will produce a single juvenile on a timer and on a 14:10 light:dark schedule which is the normal worm. Inset: micrograph (40x) of melanization spots on crayfish light cycle for summer in Boone, NC. Crayfish were provided with gills indicating past damage. Damage of this sort is possible from 2 shrimp pellets each day for nutrition. branchiobdellidan activity. Crayfish were sampled after 2 and 6 days. First, total remaining worm number was determined for crayfish stocked with worms. Candidate Gene Isolation Crayfish were snap frozen whole in liquid nitrogen and stored at Primers for amplification of the candidate immune and -80°C. At Clemson University, crayfish carapaces were removed, housekeeping genes were designed based on conserved sequences crayfish were allowed to thaw briefly, and hepatopancreas tissues identified in red swamp crayfishProcambarus ( clarkii), were removed and stored in -80°C for further analysis. After lobster (Homarus spp.), and other crustaceans. Briefly, initially harvesting the hepatopancreas, we also visually assessed any identified sequences from phylogenetically-close relatives damage to the host gills. Branchiobdellidan damage to crayfish of C. chasmodactylus were used in BLASTN searches of the gills leaves characteristic melanization spots and trunctated ends Genbank nr database. Multiple alignments of recovered sequences of gill filaments (Figure 1; Quaglio et al. 2006; Brown et al. 2012; were performed to identify highly conserved sequences across Rosewarne et al. 2012). phylogenies, and primers to those regions designed in Lasergene The day crayfish were placed in aquaria, we also field collected (DNAStar). Genes for which primers were designed include: 3 C. chasmodactylus from the same sites as experimental crayfish astacidin, 5’–GTG TTG CCC TCG TTG CTC TTA TGG–3’ and and within the same size range. We immediately froze these 3 5’–ACG TCG GCC TAC TTG CTC TTG AAC–3’; crustin, 5’– crayfish in liquid nitrogen to serve as a baseline for comparison CCC GAG GGC CCA AAC AAG–3’ and 5’–TGA ACA AGC with regards to gene expression of experimental crayfish. For GAG CCA ACA ACC TAT–3’; prophenoloxidase, 5’–CCG GGC simplicity, we refer to these crayfish in the Results and Discussion GTG GTG CTT GAC A–3’ and 5’–CAG GCC ACC CAC ACC as field references. However, it should be recognized that these CAC AGA A–3’; and GAPDH, 5’–ACA ATG AAA TGA AAC crayfish were not true controls since the experimental manipulation CAG AAA ACA–3’ and 5’–TTG CCA AGG CGG ACA GT–3’. was presence or absence of branchiobdellidans, and each of the From a single Cambarus chasmodactylus individual on ice, collected crayfish hosted C. ingens; it would be highly unusual to claw, gill, and hepatopancreas tissues were dissected and pooled, field-collectC. chasmodacyltus in this size range in the South Fork frozen with liquid nitrogen, and pulverized. Total RNA was of the New River that do not host at least one C. ingens (Brown extracted from the homogenate (Ambion RNAqueous kit), and and Creed 2004). oligo-dT primed for first strand cDNA synthesized (New England 46 Freshwater Crayfish Volume 22, Number 1

gel. DNA was column purified from gel bands (Qiagen Gel Extraction kit), ligated to pGEMT-Easy (Promega), and clones sequenced (Clemson University Genomics Institute). Sequences were analyzed and consensus sequences developed in Lasergene. BLASTN searches were performed to identify homologues, and multiple alignments and pairwise comparisons made in Lasergene. Semi-Quantitative PCR Total RNA was isolated as above from individual hepatopancreas dissected from crayfish. DNase I (Promega)-treated RNA was used to synthesize oligo-dT primed cDNA as above, and diluted to 50 µL with nuclease free water. Polymerase chain reactions were performed in 10 µL volumes, with GoTaq Green Master Mix and 333 nM forward and reverse primers and 1 µL template. Templates consisted of experimental cDNAs, pGEMT- Easy clones (positive control), DNase I-treated RNA (to verify absence of gDNA), and nuclease free water (negative control). PCR conditions consisted of an initial 3 min denaturation at 94°C followed by 35 cycles of 94°C, 60°C, 72°C (30 sec each). Products were separated by electrophoresis through 2% agarose gel, stained with ethidium bromide, and visualized by UV-light. Images were captured by digital camera and band intensity analyzed with NIH ImageJ. Pixel intensity was determined for the band in each lane and the region above and below the band. Intensity values were imported to Microsoft Excel, and background intensity (average of regions above and below bands) subtracted from band intensity. Intensity values for each experimental gene within a given cDNA pool were normalized to the housekeeping gene GAPDH amplimer (normalized value = experimental gene intensity value / GAPDH intensity value). Data Analysis We used 2-way Analysis of Variance (ANOVA) to examine expression of individual genes with treatment and sample date as factors. Although this experiment was designed to produce data in a time series, a repeated measures analysis or within-subjects design was not appropriate since there was destructive sampling on each date, and therefore no assumed autocorrelation between dates. Residuals of responses were normally distributed within tolerance, and given our balanced design and no loss of experimental units, the assumption of inequality of variance was not problematic (Box 1954).

RESULTS There were significant differences in expression of two of Figure 2. Effect of branchiobdellidans on expression of candidate genes during the 6-day experiment. Points show mean normalized number of the three candidate immune genes — astacidin and PPO — in transcripts and error bars represent +1SE. Grey lines indicate mean (solid response to the branchiobdellidan treatment. In agreement with line) and standard error for expression in field references. A) Expression our cleaning hypothesis, both genes had lower expression in for PPO. B) Astacidin. C) Crustin-2. the presence of branchiobdellidan symbionts when compared to the 0-worm controls. On both sample day-2 and day-6, PPO responded to the branchiobdellidan treatment with 3 to 4 times Biolabs Protoscript First Strand Synthesis kit). Four microliters as many transcripts in the 0-worm control as compared to the 10- of cDNA was used in a 50 µL volume PCR with GoTaq Green worm treatment (Figure 2A, Table 1); there was also a significant Master Mix (Promega) with 200 nM of forward and reverse direct effect of time P( = 0.0099) with expression increasing from primers. Cycling conditions were as follows: 3 min at 94°C; 35 day 2 to day 6, but there was not a treatment × sample interaction cycles of 30 sec at 94°C, 30 sec at 55°C, 30 sec at 72°C; 4°C. (Figure 2A, Table 1). In comparison to the Field reference crayfish, Bands of the expected size were excised from a low melt agarose expression of PPO in the 10-worm treatment was always lower, 2016 Brown et al. — Symbionts Influence Crayfish Gene Expression 47

Table 1. Results of 2-way Analysis of Variance models testing for the effects of symbiont presence on gene expression in genes associated with astacidin and prophenoloxidase. Sums of Model Effect df F-ratio P (>F) Squares Branchiobdellidans 1 0.52 5.025 0.036 Time 1 1.19 11.44 0.0030 Astacidin Branch x Time 1 0.12 1.45 0.30 Residuals 20 2.07

Branchiobdellidans 1 0.65 11.72 0.0027 Time 1 0.45 8.12 0.0099 Prophenoloxidase Branch x Time 1 0.0057 0.10 0.75 Residuals 20 1.11

Branchiobdellidans 1 0.36 0.57 0.46 Time 1 0.001 0.001 0.97 Crustin-2 Branch x Time 1 0.013 0.02 0.89 Residuals 20 12.50 while the no-worm treatment fell within the Field reference range to the melanization cascade that is a fundamental immune defense on day 2, but was higher than the Field reference on day 6 (Figure mechanism in invertebrates (Aspán et al. 1995; Cerenius and 2A). Soderhall 2004). Melanization is a key defense mechanism in organisms with chitinous exoskeletons in which damaged tissues Expression of astacidin followed a similar pattern with or foreign bodies are isolated through the melanization of tissue significant effects of both branchiobdellidans (P = 0.036) and which physically shields intrusion and actively prohibits biotic time (P = 0.0030) but not the treatment × time interaction (Figure invasion through the secondary production of toxic quinones 2B, Table 1). Like PPO, astacidin was significantly lower in the (Cerenius and Soderhall 2004). During the melanization cascade, 10-worm treatment than in the 0-worm control on both dates and prophenoloxidase is activated to phenoloxidase through cleavage expression increased from day 2 to day 6. One difference between by serine proteinases, which then catalyzes the production of astacidin and PPO was how expression compared to expression quinones with melanin as a downstream product. (Cerenius and in the field reference crayfish. Like PPO expression, astacidin Soderhall 2004). Astacidin is a relatively recently discovered expression in the 10-worm treatment was always lower than in the antimicrobial peptide isolated from the haemolymph of crayfish field reference. However, unlike with PPO, expression in the no- that inhibits growth of both Gram-positive and Gram-negative worm treatment on day 2 was also lower than the field reference, bacteria (Lee et al. 2003). Unlike PPO, the relationship between while day 6 was well within the range of the field reference. immune function and production of this peptide appears to be Crustin-2 was the only gene whose expression changed with direct and not part of a complex cascade, so that there is a direct neither treatment nor time and all observations were either within, correlation between increased transcripts and increased presence or very close to the range of the field reference values (Figure 2C). of astacidin in crayfish haemolymph (Lee et al. 2003). Similar to astacidin, crustin is an antimicrobial peptide, expressed by DISCUSSION members across Crustaceans (Smith et al. 2008). In red swamp Branchiobdellidan symbionts significantly affected the crayfish, Procambarus clarkii, crustin transcript and peptide titers expression of two immune function genes (astacidin and PPO). are upregulated upon intrahemocoelic pathogen challenge (Liu Within the context of our experiment, three results are particularly et al. 2016). To the best of our knowledge, increased expression pertinent to the ultimate interpretation of these outcomes. The first of these genes results in a general increase in immune function. result is the treatment effect that resulted in lower gene expression Astacidin and crustin are straightforward protein products of in the 10-worm treatment, the second is the significant increase expression and so there is a more direct, monotonic relationship in expression through time measured in astacidin and PPO, and between expression and the abundance of antimicrobial peptides the third is the relative level of expression with respect to the field (Lee et al. 2003). In the case of PPO, up-regulation is a precursor references. However, to clearly interpret these effects first requires to increasing activity of the phenoloxidase enzyme that catalyzes placing the results in a physiological context. What do changes in the ultimate steps of the melanization cascade (Boucias and these genes actually signify for a host crayfish immune system? Pendland 1998). Both astacidin and PPO are known to be key elements of the Given this physiological background, we can begin to invertebrate immune system. PPO is a well-described contributor interpret the results of this experiment with regard to the influence 48 Freshwater Crayfish Volume 22, Number 1 of branchiobdellidans on crayfish immune function. First, in our free controls (Brown et al. 2002; Lee et al. 2009; Brown et al. experiment there were clearly effects of the branchiobdellidan 2012; Ames et al. 2015; Thomas et al. 2016); the mechanism in C. ingens on gene expression of two immune-related genes, astacidin these cases appears to be cleaning of the host exoskeleton and, and PPO, on the host crayfish C. chasmodatcylus. In both cases, particularly, the gill filaments, through the opportunistic feeding branchiobdellidan presence led to decreased expression which was of the worms (Brown et al. 2002). While branchiobdellidans are counter to the gill damage hypothesis. The logic behind the damage known to damage the gills of their hosts (Quaglio et al. 2006; hypothesis was based on the knowledge that branchiobdellidans Brown et al. 2012; Rosewarne et al. 2012) it is likely that they can damage their hosts’ gill tissues (Quaglio et al. 2006; Rosewarne only do significant damage in high densities with the exception of et al. 2012), including the species under study (Brown et al. 2012), some species that are known gill parasites (Skelton et al. 2013). and that this damage can result in negative effects on growth of However, C. ingens is not known to be exclusively parasitic, host crayfish, including the host species in this study (Brown et and our own observations of its gut contents suggest a primarily al. 2012). This damage occurs through opportunistic feeding of predatory ecology. An additional argument for this mechanism branchiobdellidans which will scrape hosts’ gills with a pair of is that, as described in the Methods, the aquarium environment sclerotized toothlike structures (Brinkhurst and Gelder 2001). in the experiment was intentionally created to be a high-fouling In many cases, this rasping may only remove biofilm substrates environment, and positive effects of branchiobdellidan cleaning on from gills, but in other cases, entire pieces of gill filaments are crayfish are more likely to occur in higher fouling environments consumed (Rosewarne et al. 2012), creating significant damage (Lee et al. 2009). to crayfish gills that can result in significant loss of haemolymph A second piece of evidence that can help explain the results of (Quaglio et al. 2006) and potential entry of bacterial pathogens this experiment is that for astacidin and PPO, there was a significant (Cerenius and Soderhall 2004). We expected this physical damage, increase in gene expression from day-2 to day-6. This increase the associated loss of haemolymph, and the possibility of invasion occurred in both the 0-worm control and in the 10-worm treatment, by pathogens could precipitate up-regulation in the presence of indicating that this effect was independent of branchiobdellidan branchiobdellidans. Instead, expression of astacidin and PPO was activity. Again, the most likely explanation for this result is lower with the worms present. that the aquaria represented a high-fouling environment and What does decreased expression in the presence of pressure from physical fouling and potential microbial invasion branchiobdellidans indicate? There are two basic possibilities. The increased through time with increased exposure. Interestingly, first is that branchiobdellidans are actively affecting the physiology while branchiobdellidans alleviated these effects to some extent, of their hosts and inducing decreased expression of immune- they were not able to completely compensate and consequently related genes through direct chemical or genetic interaction. Such expression of astacidin and PPO increased in both the control and interactions have been described in a variety of host-symbiont treatment groups from day-2 to day-6. Effects of the MgCl2 used pairs. In some cases, selective induction of expression clearly to remove branchiobdellidans at the beginning of the experiment benefits the host and often results in host selection for very specific might have decreased microbial load on the hosts and contributed symbiont species or strains, for example for strains of Vibrio in to this pattern (Hotchkiss 1923). However, this potential effect of marine copepods (Almada and Tarrant 2016) and bacteria in Hydra MgCl2 doesn’t actually change the interpretation of the increase in (Franzenburg et al. 2013). In other cases, selective induction of expression, except that hosts might have been moving from a low expression benefits the symbiont and is cited as a general strategy to normal microbial load rather than a normal to high microbial of bacteria to circumvent insect defenses (Vallet-Gely et al. 2008). load in the 6-day experimental period. If branchiobdellidans were having these effects, the purpose would A comparison between the experimental results and field likely be to decrease the efficacy of the melanization cascade that references also supports the argument that crayfish were in a may inhibit branchiobdellidan feeding on host gills. However, stressful environment and that branchiobdellidans were able to this mechanism is not likely due to the lack of an obvious means alleviate that stress to some extent. An enlightening result is that through which branchiobdellidans can directly affect specific host for astacidin and PPO, expression in the control group largely gene expression through chemical or genetic interaction. Unlike overlapped with the field references and at least one of the two the previously cited examples, branchiobdellidans are strictly control samples fell within the range of the field reference, with ectosymbionts whose only contact with the interior of their hosts the other sample lying close to the range of the field reference. would be very limited exposure to haemolymph on damaged gill However, for astacidin and PPO, the 10-worm treatment never tissue. It is possible that branchiobdellidans may have some oral fell within the range of the field reference, indicating that secretion that interacts with host haemolymph, but to the best of branchiobdellidans were likely causing a decrease in expression our knowledge, no such secretions have been described. as opposed to the 0-worm control showing increased expression. A much more likely possibility is that, in the case of this Of course, this interpretation begs the question: If all of the field experiment, branchiobdellidans were actively alleviating stress on reference crayfish hosted branchiobdellidans, why did the field the host by removing microbial and particulate material from the references overlap with the 0-worm controls? Again, the likely crayfish exoskeleton, resulting in decreased expression of immune answer is that the aquaria represented a high-fouling environment associated genes. There is also a well-supported mechanism for and that stream dwelling crayfish rarely encounter such conditions, these effects. In multiple experiments, branchiobdellidans have at least when water is abundant and flowing as was the case when increased growth of their hosts relative to branchiobdellidan- these crayfish were collected. 2016 Brown et al. — Symbionts Influence Crayfish Gene Expression 49

Taken together, the evidence from the experiment strongly Aspán A, Huang TS, Cerenius L and Söderhäll K (1995). cDNA suggests that a) the high fouling aquarium environment was cloning of prophenoloxidase from the freshwater crayfish stressful to crayfish, and b) that branchiobdellidans were able to Pacifastacus leniusculus and its activation. Proceedings of the alleviate some, but not all, of this environmental stress. These National Academy of Sciences of the United States of America results also agree with a variety of previous work suggesting that the 92(4):939–943. crayfish-branchiobdellidan symbiosis can be a cleaning mutualism Backhed F, Ley RE, Sonnenburg JL, Peterson DA and under some environmental conditions, but that the relationship is Gordon JI (2005). Host-bacterial mutualism in the human context dependent and may shift to a commensalism or parasitism intestine. Science 307(5717):1915–1920. in other conditions (Lee et al. 2009; Brown et al. 2012; Thomas et Bauer RT (1998). Gill-cleaning mechanisms of the crayfish al. 2013). One unexpected result is that there was a strong response Procambarus clarkii (Astacidea: ): experimental of astacidin while there was no significant response ofcrustin . We testing of setobranch function. Invertebrate Biology anticipated that these two genes would have very similar responses 117(2):129–143. to treatments since they are both antimicrobial peptides that are part of the generalized immune defense. It is possible that astacidin Boucias DG and Pendland JC (1998). Insect Immune Defense may be more sensitive to environmental stimuli or have greater System, Part III: Prophenoloxidase Cascade and Post- responsiveness to particular microbial strains that were found in Attachment Processes of Phagocytosis. Pp. 499–537 Principles the experimental environment, but the exact reason for this lack of Insect Pathology. Springer US, Boston, MA, USA. of response in crustin cannot be ascertained from this experiment. Box GEP (1954). Some theorems on quadratic forms applied This study adds to a growing appreciation for the importance of in the study of analysis of variance problems, II. Effects of symbiosis, and for the range of effects that symbionts can have on inequality of variance and of correlation between errors in their hosts. It also does so with an interesting twist: it demonstrates the two-way classification. Annals of Mathematical Statistics that ectosymbionts can indirectly induce changes in gene 25(3):484–498. expression of hosts by changing the host-environment interaction. Brinkhurst RO and Gelder SR (2001). Annelida: Oligochaeta, While previous studies have demonstrated clear evidence of including Branchiobdellida. Pp. 431–463 In: Thorp JH altered gene expression in symbioses, the majority of and Covich AP, eds.). Ecology and Classification of North these results have come from endosybmionts, primarily bacteria, American Freshwater Invertebrates. Academic Press, San directly interacting with host physiology (Vallet-Gely et al. 2008; Diego, CA, USA. Franzenburg et al. 2013; Almada and Tarrant 2016). Continuing Brown B, Creed R, Skelton J, Rollins M and Farrell K investigation of these sorts of gene × environment interactions (2012). The fine line between mutualism and parasitism: mediated by symbionts should strongly contribute to our growing complex effects in a cleaning symbiosis demonstrated by knowledge regarding both symbiont effects on hosts, and how multiple field experiments.Oecologia 170(1):199–207. symbionts can modify host interactions with environment. These Brown BL and Creed RP (2004). Host preference by an studies can also be greatly enhanced through the use of systems aquatic ectosymbiotic annelid on 2 sympatric species of host like the crayfish-branchiobdellidan symbiosis. The system has a crayfishes. Journal of the North American Benthological number of advantages including ubiquity of organisms, ease of Society 23(1):90–100. manipulation of both symbionts and environmental conditions, ease of husbandry, and a rapidly growing background on the Brown BL, Creed RP and Dobson WE (2002). Branchiobdellid ecological relationships between hosts and symbionts, and we annelids and their crayfish hosts: are they engaged in a cleaning suspect that continued investigations of the interaction between symbiosis? Oecologia 132(2):250–255. gene expression, environmental conditions and symbiosis in this Carstensen DW, Sabatino M and Morellato LPC (2016). system would prove very fruitful. Modularity, pollination systems, and interaction turnover in plant-pollinator networks across space. Ecology 97(5):1298– ACKNOWLEDGMENTS 1306. We are grateful for help from Patty Whitener, Josh Lomonaco, Cerenius L and Soderhall K (2004). The prophenoloxidase- and Mark Rollins. This work was supported by funding from the activating system in invertebrates. Immunological Reviews National Science Foundation to BLB (DEB-0949780) and RPC 198:116–126. (DEB-0949823). Choi JY, Lee TW, Jeon KW and Ahn TI (1997). Evidence for symbiont-induced alteration of a host’s gene expression: LITERATURE CITED irreversible loss of SAM synthetase from Amoeba proteus. Almada AA and Tarrant AM (2016). Vibrio elicits targeted Journal of Eukaryotic Microbiology 44(5):412–419. transcriptional responses from copepod hosts. FEMS Egerton FN (2014). History of Ecological Sciences, Part 52: Microbiology Ecology 92(6):IW72–IW72. Symbiosis Studies. Bulletin of the Ecological Society of Ames CW, Helms BS and Stoeckel JA (2015). Habitat mediates America 96(1):80–139. the outcome of a cleaning symbiosis for a facultatively Farrell KJ, Creed RP and Brown BL (2014). Preventing burrowing crayfish. 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